N-terminal acetyltransferases (NATs) mediate co- and post-translational N-terminal protein acetylation to mediate essential protein functions, and their altered activities are linked to several diseases. Because of this connection of NATs to normal and abnormal cellular function, it is important to have assays available to evaluate NAT activity in vitro. This chapter outlines three in vitro assays for studying NAT activity: a sensitive radioactive method, and a scalable fluorescence-based approach and luminescence-coupled assay compatible with high-throughput screening. Using human NatB as a model NAT, we compare assay performance, highlighting differences in sensitivity, substrate requirements, and inhibitor detection. These methods enable investigations and support the development of NAT-targeted therapeutics, thus highlighting their utility in basic research and drug discovery.